Characteristic Magnetic Resonance Image Features of Acute Network Injury in Young Patients.

Cerebral infarction is known to cause secondary degeneration of the areas connected to the primarily damaged regions. This has been named as acute network injury and is usually recognized in newborns or babies by high signal intensity on diffusion-weighted imaging (DWI). In this article, we present 2 cases demonstrating several characteristics of network injury. Some features are comparable to previous studies and others are distinctive to our cases. The patients not only showed secondary injury in the thorough pyramidal tract along the longitudinal extensions of neural tracts as expected but also followed transverse connections to reach the contralateral hemisphere. The location of network injury varied according to the initial lesion and projected in an omnidirectional manner as long as the brain parts are interconnected. In addition, the cases well demonstrated the temporal changes on brain imaging. Network injury appeared on DWI around a week after major damage and then subsequently disappeared. The overall process of appearance to disappearance was completed within 2 weeks from the symptom onset. As ominous neurological outcomes are thought to be related to acute network injuries, a comprehensive understanding of the phenomenon is pivotal in improving diagnosis and management.

Joint association of aerobic physical activity and muscle-strengthening activities with metabolic syndrome : the Korean National Health and Nutrition Examination Survey 2014-2015.

OBJECTIVES: The study aimed to examine whether simultaneously meeting the combined guidelines of accelerometer-assessed moderate to vigorous physical activity (MVPA) and self-reported muscle-strengthening activity (MSA) was associated with lower odds of metabolic syndrome (MetS) than meeting neither or 1 of the guidelines among the Koreans. METHODS: This cross-sectional analysis included 1,355 participants from the Korea National Health and Nutrition Examination Survey (2014-2015). Logistic regression was used to analyze the associations across groups of MVPA-MSA guideline adherence (meeting neither [reference]; meeting MVPA only; meeting MSA only; meeting both MVPS and MSA) with MetS components (abdominal obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol [HDL-C], hypertension, and hyperglycemia). The odds ratios (ORs) were adjusted for covariates (e.g., sex, age, body mass index, and accelerometer wearing time). RESULTS: MSA only significantly reduced the OR for abdominal obesity (OR, 0.34; 95% confidence interval [CI], 0.13 to 0.91). Meeting both MVPA and MSA reduced the OR for hypertriglyceridemia (OR, 0.59; 95% CI, 0.39 to 0.88) and low HDL-C (OR, 0.46; 95% CI, 0.31 to 0.68). Compared to meeting neither, MVPA only (OR, 0.63; 95% CI, 0.44 to 0.89) and both MVPA and MSA (OR, 0.46; 95% CI, 0.28 to 0.76) significantly reduced the OR for MetS. CONCLUSIONS: Combined MVPA-MSA was more beneficially associated with MetS prevalence than MVPA only and MSA only. Considering that more than 85% of Korean adults do not meet both the MVPA and MSA guidelines, public health actions to promote adherence should be supported.

Stepwise Observation of Iodine Diffusion in a Flexible Coordination Network Having Dual Interactive Sites.

We created dual interactive sites in a porous coordination network using a CuI cluster and a rotation-restricted ligand, tetra(3-pyridyl)phenylmethane (3-TPPM). The dual interactive sites of iodide and Cu ions can adsorb I2 via four-step processes including two chemisorption processes. Initially, one I2 molecule was physisorbed in a pore and successively chemisorbed on iodide sites of the pore surface, and then the next I2 molecule was physisorbed and chemisorbed on Cu ions to form a cross-linked network. We revealed the four-step I2 diffusion process by single-crystal X-ray structure determination and spectroscopic kinetic analysis.

Accelerometer-measured stepping cadence patterns in Korean adults: an analysis of data from the 2014-2015 Korea National Health and Nutrition Examination Survey.

OBJECTIVES: The aim of this study was to identify the stepping cadence patterns in Korean adults by using objectively measured accelerometer data to analyze the time spent in each cadence category by sex and age. METHODS: During the 2014-2015 Korean National Health and Nutrition Examination Survey, 1,703 males and females aged 19-64 years provided at least 1 valid day of data (wearing an accelerometer ≥ 10 hr/d). The mean cumulative time and percentage per day in 8 cadence categories (0, 1-19, 20-29, 30-59, 60-79, 80-99, 100-119, and ≥ 120 steps/min) by sex and age group were calculated. RESULTS: Cumulative time and percentage per day decreased across the incremental cadence categories. Participants spent 360.08± 2.56 min/d in the non-movement cadence category and 361.50± 2.28 min/d in the incidental movement cadence category. However, they spent only about 18.1 min/d (2.1%) at ≥ 100 steps/min. Males spent significantly more time in the cadence categories of sporadic movement, purposeful steps, slow walking, and medium walking, but the other categories, except for brisk walking, had higher values in females (p< 0.001). The older age group spent less time in non-movement cadence categories, and the youngest and oldest groups spent more time at a higher cadence (≥ 100 steps/min) than the other age groups. Similar patterns were found in a subgroup analysis by sex. CONCLUSIONS: Korean adults spent most of their time at a low cadence and only a few minutes at a high cadence (≥100 steps/min); this trend was consistent across sex and age groups.

Relationship of Physical Activity and Sedentary Time with Metabolic Health in Children and Adolescents Measured by Accelerometer: A Narrative Review.

The purpose of this study was to summarize the associations of physical activity (PA) and sedentary time (SED) with metabolic health and examine the effects of time reallocation on metabolic health in adolescents using accelerometer data. A literature search was conducted using PubMed, ScienceDirect, Web of Science, Cochran Library, and Google Scholar, and 27 articles were reviewed. Recent research generally confirms the associations of PA and SED with metabolic health. High PA levels and low SED levels had a positive relationship with metabolic health. Moreover, reallocating 10 min of daily SED to PA was associated with better metabolic health indicators. These results were stronger for moderate-to-vigorous physical activity than for light intensity PA. Thus, efforts to convert SED into PA of at least moderate intensity appear to be an effective strategy to prevent metabolic disease development in children and adolescents. However, some of the associations between PA and metabolic health indicators were inconsistent, depending on age, obesity degree, and PA intensity. Additionally, various accelerometer data collection and processing criteria impact the interpretation of the results. Therefore, consistent accelerometer data collection and analysis methods are needed in future studies. Further, intervention studies are required to verify the causality and effectiveness of the isotemporal substitution model.

Development of the Parental Questionnaire for Cerebral Visual Impairment in Children Younger than 72 Months.

BACKGROUND AND PURPOSE: Cerebral visual impairment (CVI) is an underdiagnosed condition in children, and its assessment tools have focused on older children. We aimed to develop a parental questionnaire for cerebral visual impairment (PQCVI) for screening CVI in young children. METHODS: The PQCVI comprised 23 questions based on a modified version of Houliston and Dutton's questionnaire for older children. The PQCVI with neurocognitive function tests was applied to 201 child-parent pairs with typically developing children younger than 72 months (age 32.4±20.1 months, mean±standard deviation). The children were classified into six age groups. The normative data, cutoff scores, and internal reliability were assessed and item analysis was performed. We referred to the total score for all questions as the cerebral visual function (CVF) score. RESULTS: The normative data showed that the CVF score and the scores corresponding to ventral-stream and dorsal-stream visual functions plausibly increased with age. The scores rapidly reached 90% of their maximum values up to the age of 36 months, after which they increased slowly. Cronbach's alpha for all questions across all age groups was 0.97, showing excellent consistency. The item difficulty and item discrimination coefficients showed that the questions were generally adequate for this age stage. CONCLUSIONS: The PQCVI items produced reliable responses in children younger than 72 months. The rapid increase in scores before the age of 3 years supports the importance of early identification of CVI. Following additional clinical verification, the PQCVI may be useful for CVI screening.

Factors associated with stigma and depressive symptoms in family members of patients with epilepsy.

PURPOSE: Literature regarding family stigma related to epilepsy is scarce. This study investigated the prevalence of family stigma and depressive symptoms and the associated factors among the family members of patients with epilepsy. METHODS: In a cross-sectional study, Stigma Scale-Revised score ≥ 4 and Patient Health Questionnaire-9 score ≥ 10 were considered indicative of moderate-to-severe stigma and depressive symptoms, respectively. Stepwise logistic regression analyses were performed. RESULTS: Of the 482 family members, a mean age was 47.1 ±â€¯9.4 years, and 73.4% were female. Of the patients, a mean age was 25.5 ±â€¯16.7 years, and 45.0% were female. Idiopathic generalized epilepsy and focal epilepsy were noted in 22.4% and 65.6% of patients, respectively. Family stigma and depressive symptoms were noted in 10.0% and 11.2% of family members, respectively. Family stigma was significantly associated with high seizure frequency and being a sibling or offspring of a patient independent of their depressive symptoms. By contrast, depressive symptoms in family members were significantly associated with polytherapy, being parents of a patient, and neurological comorbidities independent of family stigma. In a subset of patients and their family, patients had higher proportion of stigma and depressive symptoms than their family. Depressive symptoms and stigma among patients were significantly correlated with those among parents, but not spouse. CONCLUSION: Family stigma is common in families with epilepsy and is closely related to depressive symptoms. Frequent seizures, polytherapy, neurological comorbidities, and the relationship to a patient may be factors that are independently associated with family stigma and depressive symptoms in family members.

Does the new Korean term for epilepsy reduce the stigma for Korean adults with epilepsy?

PURPOSE: The purpose of this study was to evaluate differences in stigma, disclosure management of epilepsy, and knowledge about epilepsy between patients with epilepsy who recognized and did not recognize the new Korean term for epilepsy. METHODS: This was a cross-sectional, multicenter study. The Stigma Scale-Revised, the Disclosure Management Scale, the Patient Health Questionnaire-9, and a questionnaire assessing knowledge about epilepsy were used. The set of questionnaires had two versions, using either the old or new name for epilepsy. Multivariate logistic regression analyses were used. RESULTS: A total of 341 patients with epilepsy and 509 family members were recruited. Approximately 62% of patients felt some degree of epilepsy-related stigma. Mild stigma, severe concealment of epilepsy diagnosis, and increased knowledge about epilepsy were independently identified as factors associated with recognition of the new term in patients. Recognition of the new term was more prevalent in patients and family members with higher education, female family members, and family members having patients with younger age at seizure onset and shorter duration of epilepsy. There were no significant differences between the two types of questionnaires. About 81% of patients and 93% of family members had a positive attitude about renaming epilepsy. CONCLUSION: The use of the new Korean term for epilepsy (cerebroelectric disorder) increased knowledge about epilepsy but did not reduce stigma and concealment of epilepsy diagnosis in Korean adults with epilepsy. Higher education may be an important factor for knowing the new term in patients and family members.

Effect of acute intradialytic aerobic and resistance exercise on one-day blood pressure in patients undergoing hemodialysis: a pilot study.

BACKGROUND: Hypertension and intradialytic hypotension are independent risk factors for mortality in hemodialysis patients. We hypothesized that intradialytic exercise would increase blood pressure (BP) during dialysis and decrease it during the postdialytic period. The present study aimed to investigate the effect of acute intradialytic exercise on BP both during dialysis and for 20 hours postdialysis, and to detect any differences in effects of aerobic exercise (AE), resistance exercise (RE), and usual care (UC-the control condition). METHODS: Eleven patients undergoing maintenance hemodialysis performed two complete sets of AE or RE, with a 1-hour rest between the sets. The patients performed AE, RE and UC over three consecutive weeks at 7-day intervals. Intradialytic BP was measured using an oscillometric BP monitor (N.=11), and ambulatory BP was measured for 20 hours after each dialysis session using an ambulatory BP monitor (N.=8). RESULTS: The mean BP of the patients in the AE and RE interventions increased during exercise (P<0.05), with the exception of the first set of AE. However, only RE increased BP significantly compared with UC (P<0.05). Following dialysis, daytime ambulatory BP was significantly lower after AE and RE than after UC (P<0.05). CONCLUSIONS: Acute intradialytic exercise interventions are effective in increasing BP during dialysis and decreasing daytime ambulatory BP after dialysis. Longer observation periods and larger sample sizes will be needed to confirm our findings. Also further studies should be performed on patients prone to intradialytic hypotension.

Combined exercise improves gastrointestinal motility in psychiatric in patients.

AIM: To examine the effect of combined exercise on colonic transit time (CTT) in admitted psychiatric patients. METHODS: Over a 6-mo period, consecutive in patients with mental illness were recruited from the Somang Hospital Psychiatry Unit. A combined exercise program that included 60 min per day of exercise 3 d per week for 12 wk was performed. Physical fitness and CTT of the patients were measured twice before and twice after the exercise program. CTT was measured using a multiple marker technique with a radio-opaque marker. Changes in the exercising patients' CTT and weight-, cardiovascular- and fitness-related parameters were statistically assessed. RESULTS: After the 12-wk combined exercise intervention, decreased intestinal transit time was observed in all CTTs of the exercise group, including the right CTT (exercise: 15.6 ± 15.2 vs 9.2 ± 11.9, control: 13.1 ± 10.4 vs 10.9 ± 18.7), left CTT (exercise: 19.7 ± 23.5 vs 10.4 ± 13.2, control: 19.2 ± 19.0 vs 16.9 ± 19.8), recto-sigmoid CTT (exercise: 14.3 ± 16.7 vs 6.7 ± 7.9, control: 15.0 ± 14.4 vs 19.3 ± 30.3), and total colonic transit time (TCTT) (exercise: 50.2 ± 38.1 vs 27.1 ± 28.0, control: 47.4 ± 34.6 vs 47.3 ± 47.3). After the 12-wk combined exercise period, TCTT was significantly shortened in the exercise group compared with that in the control group. In addition to eating habits, water intake, and fiber intake, the increased physical activity level as a result of the 12-wk combined exercise program reduced the CTT. CONCLUSION: The CTT of the psychiatric patients was reduced due to increased physical activity via a 12-wk combined exercise program.

The effect of ephedrine on intraoperative hypothermia.

BACKGROUND: Prevention of intraoperative hypothermia has become a standard of operative care. Since ephedrine has a thermogenic effect and it is frequently used to treat hypotension during anesthesia, this study was designed to determine the effect of ephedrine on intraoperative hypothermia of patients who are undergoing spine surgery. METHODS: Twenty-four patients were randomly divided to receive an ephedrine (the ephedrine group, n = 12) or normal saline (the control group, n = 12) infusion for 2 h. The esophageal temperature (the core temperature), the index finger temperature (the peripheral temperature) and the hemodynamic variables such as the mean blood pressure and heart rate were measured every 15 minutes after the intubation. RESULTS: At the end of the study period, the esophageal temperature and hemodynamic variables were significantly decreased in the control group, whereas those in the ephedrine group were stably maintained. The index finger temperature was significantly lower in the ephedrine group compared to that in the control group, suggesting the prevention of core-to-peripheral redistribution of the heat as the cause of temperature maintenance. CONCLUSIONS: An intraoperative infusion of ephedrine minimized the decrease of the core temperature and it stably maintained the hemodynamic variables during spine surgery with the patient under general anesthesia.

Caudal analgesia reduces the sevoflurane requirement for LMA removal in anesthetized children.

BACKGROUND: An anesthetic state can reduce adverse airway reaction during laryngeal mask airway (LMA) removal in children. However, the anesthetic state has risks of upper airway obstruction or delayed emergence; so possibly less anesthetic depth is advisable. Caudal analgesia reduces the requirement of anesthetic agents for sedation or anesthesia; it is expected to reduce the sevoflurane requirement for LMA removal. Therefore, we determined the EC(50) of sevoflurane for LMA removal with caudal analgesia and compared that to the EC(50) without caudal analgesia. METHODS: Forty-three unpremedicated children aged 1 to 6 yr were enrolled. They were allocated to receive or not to receive caudal block according to their parents' consent. General anesthesia were induced and maintained with sevoflurane and oxygen in air. EC(50) of sevoflurane for a smooth LMA removal with and without caudal analgesia were estimated by the Dixon up-and-down method. The LMA was removed when predetermined end-tidal sevoflurane concentration was achieved, and the sevoflurane concentration of a subsequent patient was determined by the success or failure of the previous patient with 0.2% as the step size; success was defined by the absence of an adverse airway reaction during and after LMA removal. EC(50) of sevoflurane with caudal block, and that without caudal block, were compared by a rank-sum test. RESULTS: The EC(50) of sevoflurane to achieve successful LMA removal in children with caudal block was 1.47%; 1.81% without caudal block. The EC(50) were significantly different between the two groups (P < 0.001). CONCLUSIONS: Caudal analgesia significantly reduced the sevoflurane concentration for a smooth LMA removal in anesthetized children.

[A case of partial trisomy 15q25.3-qter].

A 15q25-qter partial trisomy characterized by pre or postnatal overgrowth, tall stature, macrocephaly and craniosynostosis has rarely been reported. The cause of overgrowth has been thought to be the triplication of the insulin-like growth factor 1 receptor (IGF1R) gene located on the 15q26.3. We report a patient with partial trisomy 15q25.3-qter showing mental retardation, developmental delay, macrocephaly, long narrow face, ptosis, high palate arch, scoliosis, clinodactyly and overgrowth. Additional material located on terminal 2q was found in karyotyping analysis. In bacterial artificial chromosome (BAC) clone-based-array comparative genomic hybridization (aCGH) analysis, a gain of 31 clones on 15q25.3-qter and a loss of 2 clones on 2q37.3 were observed. An extra copy of IGF1R gene was observed on derivative chromosome 2 in FISH analysis. In conclusion, the patient was diagnosed to have de novo 46,XX,der(2)t(2;15)(q37.3;q25.3) chromosome complement. Adequate genetic counseling and regular follow-ups would be needed for the patient.

Lateral segregation of anionic phospholipids in model membranes induced by cytochrome P450 2B1: bi-directional coupling between CYP2B1 and anionic phospholipid.

The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.

The effects on cognitive function and behavioral problems of topiramate compared to carbamazepine as monotherapy for children with benign rolandic epilepsy.

PURPOSE: To evaluate the cognitive and behavioral effects of topiramate (TPM) versus carbamazepine (CBZ) using efficacious doses of each drug as monotherapy for children with benign rolandic epilepsy. METHODS: A multicenter, randomized, open-label, observer-blinded, parallel-group clinical trial was conducted. TPM was introduced at a dose of 12.5 mg/day with the minimum target dose of 50 mg/day in patients <30 kg and 75 mg/day in patients >30 kg over 4 weeks. CBZ was started at a dose of 10 mg/kg/day with the minimum target dose of 20 mg/kg/day over 4 weeks. Additional individual escalation was allowed up to a maximum target dose. The primary study end point was change on a neuropsychological test battery after 28 weeks of treatment. RESULTS: Neuropsychological data were available for 88 patients (45 patients for TPM and 43 patients for CBZ). Of the cognitive variables measured, arithmetic showed significant worsening in TPM (p = 0.037). An additional test, for maze, also showed a significantly greater improvement for CBZ (p = 0.026). Of behavioral variables, no significant changes were found but the scores had a negative trend for the TPM. When 30 patients on the minimum target dose for TPM were compared to 40 patients treated with minimum target CBZ, there was no significant worsening of cognitive and behavioral effects in the TPM. CONCLUSION: The pattern of neuropsychometric changes with TPM seemed to be slightly worse overall than CBZ. However, outcome with the minimum target dose did not differ significantly in comparisons between the treatment groups.

Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

Inhibition of human cytochrome P450 3A4 activity by zinc(II) ion.

Effects of Zn2+ on the activity and conformation of cytochorome P450 3A4 (CYP3A4) were investigated. Zn2+ specifically inhibited the testosterone 6beta-hydroxylation activity of CYP3A4 with an IC50 value of 27 microM. Zn2+ inhibited the CO-binding spectra of CYP3A4 reduced by NADPH-cytochrome P450 reductase (CPR) and NADPH only in the presence of b5. Zn2+-induced conformational changes of CYP3A4 were monitored by CD and intrinsic fluorescence. Zn2+ showed no significant effects on the activity of CYP3A4 supported by tert-butyl hydroperoxide, an oxygen surrogate, and on the reduction of b5 by CPR and NADPH. These results suggest that the inhibitory effects of Zn2+ come from preventing the stimulation of b5 on CYP3A4 activity.

Non-specific inhibition of human cytochrome P450-catalyzed reactions by hemin.

Hemin, a stable form of heme, is known to have an antimutagenic effect. Inhibitory effects of hemin on the cytochrome P450 (CYP)-catalyzed reactions of human liver microsomes and reconstituted systems containing purified CYP and NADPH-cytochrome P450 reductase (NPR) were seen. Hemin non-specifically inhibited all of the microsomal CYP activities examined. Hemin also inhibited 7-ethoxyresorufin O-deethylation, 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin O-demethylation, and testosterone 6beta-hydroxylation catalyzed by purified CYPs 1A2, 2D6, and 3A4, with IC50 values of 27, 19, and 2.4 microM, respectively. Hemin also inhibited reduction of cytochrome c and ferricyanide by NPR, as much as 47%. Spectrally detectable CYP was destroyed in human liver microsomes and in a reconstituted system in the presence of hemin and an NADPH-generating system. We propose that the antimutagenic effect of hemin might be due to inhibition of CYP and NPR enzymes involved in the bioactivation of mutagens.

Differential effect of copper (II) on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase: inhibition of cytochrome P450-catalyzed reactions by copper (II) ion.

Inhibitory effects of Cu(2+) on the cytochrome P450 (P450)-catalyzed reactions of liver microsomes and reconstituted systems containing purified P450 and NADPH-P450 reductase (NPR) were seen. However, Zn(2+), Mg(2+), Mn(2+), Ca(2+), and Co(2+) had no apparent effects on the activities of microsomal P450s. Cu(2+) inhibited the reactions catalyzed by purified P450s 1A2 and 3A4 with IC(50) values of 5.7 and 8.4 microM, respectively. Cu(2+) also inhibited reduction of cytochrome c by NPR (IC(50) value of 5.8 microM). Copper caused a decrease in semiquinone levels of NPR, although it did not disturb the rate of formation of semiquinone. P450 reactions supported by an oxygen surrogate, tert-butyl hydroperoxide, instead of NPR and NADPH, were inhibited by the presence of Cu(2+). The results indicate that Cu(2+) inhibits the P450-catalyzed reactions by affecting both P450s and NPR. It was also found that the inhibition of catalytic activities of P450s by Cu(2+) involves overall conformational changes of P450s and NPR, investigated by CD and intrinsic fluorescence spectroscopy. These results suggest that the inhibitory effect of Cu(2+) on the P450-catalyzed reactions may come from the inability of an efficient electron transfer from NPR to P450 and also the dysfunctional conformation of NPR and P450.

Development of peptide substrates for trypsin based on monomer/excimer fluorescence of pyrene.

An assay using fluorogenic peptides based on the monomer/excimer fluorescence features of pyrene was developed to measure the proteolytic activity of trypsin, a serine protease. Two pyrene moieties were incorporated into the respective N- and C-terminus of the peptides as (pyrene)-C-Xaa-C-(pyrene), where Xaa represents amino acid residues of 5-, 6-, 7-, or 8-mer containing the cleavage site of trypsin. The proteolytic cleavage of the substrates led to an increase in monomer fluorescence and a decrease in excimer fluorescence of pyrene. Kinetic parameters (k(cat) and K(m)) for the enzymatic hydrolysis of the substrates were successfully determined. The parameters are dependent on the chain length of the substrate and optimal catalytic activity was obtained with substrates that consisted of 9 or 10 amino acid residues. The present assay system is sensitive and the preparation of the substrate is very simple. We suggest that this method may be suitable for high-throughput screening and also applicable to the characterization of other proteases.